Glycosylation and carbohydrate recognition are integral and essential aspects of eukaryotic biology. The array of glycans present in an organism (the glycome) is a dynamic property depending on many features including cellular localization and temporal state, and may be perturbed in disease states such as cancer, or exploited for adhesion by pathogens. Variations in glycosylation can serve not only as disease biomarkers, but can also impact the pharmacological properties of recombinant therapeutic biologics. Glycan heterogeneity can impact batch-to-batch consistency, immunogenicity, pharmacokinetics, activity, and clearance. Unlike the case of proteins and nucleic acids, the sequencing and structural characterization of glycans is a laborious multi-step process, typically requiring sample enrichment, enzyme digestion, and mass-spectrometric analysis, a process which is not amenable to real-time monitoring. More than two thirds of therapeutic biologics are glycosylated, and batch acceptance requires that the glycosylation patterns fall within set limits.
The exploitation of glycans as biomarkers in diagnostic and therapeutic applications is hindered by difficulties in generating highly specific detection reagents (Kuzmanov et al., BMC medicine 11, 31 (2013)), which is not unexpected given the immense diversity of glycan structure (Cummings, Molecular BioSystems 5, 1087-1104 (2009)). Glycans are recognized by several classes of proteins, including lectins, antibodies, and enzymes. Lectins typically display low affinities (mM to μM) and broad or context-dependent glycan recognition (Debray et al., Eur J Biochem 117, 41-55 (1981); Liener et al., The Lectins: properties, functions, and applications in biology and medicine (Academic Press, Orlando; 1986); Bertozzi & Kiessling, Science (New York, N.Y.) 291, 2357-2364 (2001)), yet despite these issues, lectin affinity chromatography is the most widely applied technique for the isolation and enrichment of glycans and glycoconjugates. Anti-carbohydrate antibodies may exhibit improved affinity compared to lectins, and may be highly specific for particular glycans, but they can be difficult to generate given that carbohydrates are poor immunogens in general. Glycan-processing enzymes are often very selective with regard to substrate structure, reflecting their essential role in glycan processing. Site directed mutagenesis has been employed to generate inactive mutants, facilitating the characterization of substrate specificity (Rao et al., Protein Sci 8, 2338-2346 (1999)).